Modulation of Gankyrin expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of Gankyrin. The compositions comprise oligonucleotides, targeted to nucleic acid encoding Gankyrin. Methods of using these compounds for modulation of Gankyrin expression and for diagnosis and treatment of disease associated with expression of Gankyrin are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of Gankyrin. In particular, this inventionrelates to compounds, particularly oligonucleotide compounds, which, inpreferred embodiments, hybridize with nucleic acid molecules encodingGankyrin. Such compounds are shown herein to modulate the expression ofGankyrin.

BACKGROUND OF THE INVENTION

[0002] The degradation of proteins which are no longer needed in a cellis an important regulatory mechanism for cellular processes such as thecell cycle, transcription, and signal transduction. The 26S proteasomeis responsible for most of the non-lysosomal degradation ofintracellular proteins. Many proteins that are targeted for destructionby the 26S proteasome bear a polyubiquitin chain on lysine residueswhich acts a signal for protein destruction, although ubiquitination isnot a prerequisite for degradation by the proteasome. The 26S proteasomeis comprised of two major subcomplexes, the 20S proteolytic core and the19S regulatory complex, which are comprised of 28 and 17 proteinsubunits, respectively (Ferrell et al., Trends Biochem. Sci., 2000, 25,83-88).

[0003] The 19S regulatory core is thought to have several biochemicalfunctions. First, it recognizes ubiquitinated substrates. Second, it isthought to have isopeptidase activity to cleave the polyubiquitin chainsinto monomers for reuse. Third, binding of 19S to 20S is thought to openthe narrow pore of 20S to allow access to the proteolytic site. Finally,it is postulated that 19S functions as a reverse chaperone to denaturethe proteins allowing the unfolded protein to enter the proteolyticcompartment of the 20S core (Ferrell et al., Trends Biochem. Sci., 2000,25, 83-88).

[0004] The 19S regulatory complex is composed of 17 protein subunits.Gankyrin, a 28 kDa protein, (also known as p28, Proteosome (prosome,macropain) 26S subunit, non-ATPase, 10, and PSMD10) was identified asone subunit of this 19S regulatory complex. Gankyrin was isolated frombovine erythrocytes and later cloned using a cDNA library prepared fromhuman fibrosarcoma cells and lymphoblastoma cells (Hori et al., Gene,1998, 216, 113-122). Gankyrin contains 5 ankyrin repeats, a commonprotein sequence motif present in a wide variety of proteins, which isinvolved in mediating protein-protein interactions (Hori et al., Gene,1998, 216, 113-122; Sedgwick and Smerdon, Trends Biochem. Sci., 1999,24, 311-316).

[0005] The role of Gankyrin in the proteasome and as an oncogene isunder investigation. The ankyrin repeats of Gankyrin may function as ascavenger, binding target proteins that are destined to be degraded(Dawson et al., Mol. Biol. Rep., 1997, 24, 39-44). The 19S regulatorycomplex contains several ATPase subuits, and Gankyrin associates withthe S6 ATPase, suggesting that Gankyrin may block or stimulate thedegradation of proteins (Dawson et al., J. Biol. Chem., 2002, 277(13),10893-902). In precipitates from cultured cells, the association ofGankyrin and the S6 ATPase was observed as a complex that also containedcyclin D-dependent kinase (CDK4), suggesting a role for Gankyrincomplexes in the oncogenesis mediated by CDK4 (Dawson et al., J. Biol.Chem., 2002, 277(13), 10893-902).

[0006] The importance of Gankyrin in hepatocarcinogenesis has beendemonstrated in a rodent model. Retinoblastoma protein (Rb) is atumor-supressor gene that can suppress cell proliferation in the liverof nude mice, and its inactivation might be involved in the progressionof human carcinogenesis. Gankyrin binds Rb and increases thephosphorylation and degradation of Rb in vitro and in vivo, leading toanchorage-independent cell growth. Gankyrin is commonly overexpressed inhepatoma, and when overexpressed, Gankyrin transforms NIH3T3 cells.Inoculation of nude mice with these cells produced tumors anddemonstrated that Gankyrin is oncogenic (Higashitsuji et al., Nat. Med.,2000, 6, 96-99).

[0007] Furthermore, sequential changes in Rb and Gankyrin expressionobserved during various stages of hepatocarcinogenesis in Fischer ratsindicate that Gankyrin expression induced in liver fibrosis acceleratedthe degradation of Rb during liver cirrhosis (Park et al., Mol.Carcinog., 2001, 30, 138-150).

[0008] Currently, there are no known therapeutic agents whicheffectively inhibit the synthesis of Gankyrin and to date, no inhibitorshave been reported which modulate Gankyrin function. Consequently, thereremains a long felt need for agents capable of effectively inhibitingGankyrin function.

[0009] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of Gankyrin expression.

[0010] The present invention provides compositions and methods formodulating Gankyrin expression.

SUMMARY OF THE INVENTION

[0011] The present invention is directed to compounds, especiallynucleic acid and nucleic acid-like oligomers, which are targeted to anucleic acid encoding Gankyrin, and which modulate the expression ofGankyrin. Pharmaceutical and other compositions comprising the compoundsof the invention are also provided. Further provided are methods ofscreening for modulators of Gankyrin and methods of modulating theexpression of Gankyrin in cells, tissues or animals comprisingcontacting said cells, tissues or animals with one or more of thecompounds or compositions of the invention. Methods of treating ananimal, particularly a human, suspected of having or being prone to adisease or condition associated with expression of Gankyrin are also setforth herein. Such methods comprise administering a therapeutically orprophylactically effective amount of one or more of the compounds orcompositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0012] A. Overview of the Invention

[0013] The present invention employs compounds, preferablyoligonucleotides and similar species for use in modulating the functionor effect of nucleic acid molecules encoding Gankyrin. This isaccomplished by providing oligonucleotides which specifically hybridizewith one or more nucleic acid molecules encoding Gankyrin. As usedherein, the terms “target nucleic acid” and “nucleic acid moleculeencoding Gankyrin” have been used for convenience to encompass DNAencoding Gankyrin, RNA (including pre-mRNA and mRNA or portions thereof)transcribed from such DNA, and also cDNA derived from such RNA. Thehybridization of a compound of this invention with its target nucleicacid is generally referred to as “antisense”. Consequently, thepreferred mechanism believed to be included in the practice of somepreferred embodiments of the invention is referred to herein as“antisense inhibition.” Such antisense inhibition is typically basedupon hydrogen bonding-based hybridization of oligonucleotide strands orsegments such that at least one strand or segment is cleaved, degraded,or otherwise rendered inoperable. In this regard, it is presentlypreferred to target specific nucleic acid molecules and their functionsfor such antisense inhibition.

[0014] The functions of DNA to be interfered with can includereplication and transcription. Replication and transcription, forexample, can be from an endogenous cellular template, a vector, aplasmid construct or otherwise. The functions of RNA to be interferedwith can include functions such as translocation of the RNA to a site ofprotein translation, translocation of the RNA to sites within the cellwhich are distant from the site of RNA synthesis, translation of proteinfrom the RNA, splicing of the RNA to yield one or more RNA species, andcatalytic activity or complex formation involving the RNA which may beengaged in or facilitated by the RNA. One preferred result of suchinterference with target nucleic acid function is modulation of theexpression of Gankyrin. In the context of the present invention,“modulation” and “modulation of expression” mean either an increase(stimulation) or a decrease (inhibition) in the amount or levels of anucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition isoften the preferred form of modulation of expression and mRNA is often apreferred target nucleic acid.

[0015] In the context of this invention, “hybridization” means thepairing of complementary strands of oligomeric compounds. In the presentinvention, the preferred mechanism of pairing involves hydrogen bonding,which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogenbonding, between complementary nucleoside or nucleotide bases(nucleobases) of the strands of oligomeric compounds. For example,adenine and thymine are complementary nucleobases which pair through theformation of hydrogen bonds. Hybridization can occur under varyingcircumstances.

[0016] An antisense compound is specifically hybridizable when bindingof the compound to the target nucleic acid interferes with the normalfunction of the target nucleic acid to cause a loss of activity, andthere is a sufficient degree of complementarity to avoid non-specificbinding of the antisense compound to non-target nucleic acid sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

[0017] In the present invention the phrase “stringent hybridizationconditions” or “stringent conditions” refers to conditions under which acompound of the invention will hybridize to its target sequence, but toa minimal number of other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances andin the context of this invention, “stringent conditions” under whicholigomeric compounds hybridize to a target sequence are determined bythe nature and composition of the oligomeric compounds and the assays inwhich they are being investigated.

[0018] “Complementary,” as used herein, refers to the capacity forprecise pairing between two nucleobases of an oligomeric compound. Forexample, if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

[0019] It is understood in the art that the sequence of an antisensecompound need not be 100% complementary to that of its target nucleicacid to be specifically hybridizable. Moreover, an oligonucleotide mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure or hairpin structure). It is preferred that the antisensecompounds of the present invention comprise at least 70% sequencecomplementarity to a target region within the target nucleic acid, morepreferably that they comprise 90% sequence complementarity and even morepreferably comprise 95% sequence complementarity to the target regionwithin the target nucleic acid sequence to which they are targeted. Forexample, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

[0020] B. Compounds of the Invention

[0021] According to the present invention, compounds include antisenseoligomeric compounds, antisense oligonucleotides, ribozymes, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds which hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, circular orhairpin oligomeric compounds and may contain structural elements such asinternal or terminal bulges or loops. Once introduced to a system, thecompounds of the invention may elicit the action of one or more enzymesor structural proteins to effect modification of the target nucleicacid. One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

[0022] While the preferred form of antisense compound is asingle-stranded antisense oligonucleotide, in many species theintroduction of double-stranded structures, such as double-stranded RNA(dsRNA) molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

[0023] The first evidence that dsRNA could lead to gene silencing inanimals came in 1995 from work in the nematode, Caenorhabditis elegans(Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. haveshown that the primary interference effects of dsRNA areposttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA,1998, 95, 15502-15507). The posttranscriptional antisense mechanismdefined in Caenorhabditis elegans resulting from exposure todouble-stranded RNA (dsRNA) has since been designated RNA interference(RNAi). This term has been generalized to mean antisense-mediated genesilencing involving the introduction of dsRNA leading to thesequence-specific reduction of endogenous targeted mRNA levels (Fire etal., Nature, 1998, 391, 806-811). Recently, it has been shown that itis, in fact, the single-stranded RNA oligomers of antisense polarity ofthe dsRNAs which are the potent inducers of RNAi (Tijsterman et al.,Science, 2002, 295, 694-697).

[0024] In the context of this invention, the term “oligomeric compound”refers to a polymer or oligomer comprising a plurality of monomericunits. In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologsthereof. This term includes oligonucleotides composed of naturallyoccurring nucleobases, sugars and covalent internucleoside (backbone)linkages as well as oligonucleotides having non-naturally occurringportions which function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for a target nucleic acid and increased stability inthe presence of nucleases.

[0025] While oligonucleotides are a preferred form of the compounds ofthis invention, the present invention comprehends other families ofcompounds as well, including but not limited to oligonucleotide analogsand mimetics such as those described herein.

[0026] The compounds in accordance with this invention preferablycomprise from about 8 to about 80 nucleobases (i.e. from about 8 toabout 80 linked nucleosides). One of ordinary skill in the art willappreciate that the invention embodies compounds of 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases inlength.

[0027] In one preferred embodiment, the compounds of the invention are12 to 50 nucleobases in length. One having ordinary skill in the artwill appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleobases in length.

[0028] In another preferred embodiment, the compounds of the inventionare 15 to 30 nucleobases in length. One having ordinary skill in the artwill appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

[0029] Particularly preferred compounds are oligonucleotides from about12 to about 50 nucleobases, even more preferably those comprising fromabout 15 to about 30 nucleobases.

[0030] Antisense compounds 8-80 nucleobases in length comprising astretch of at least eight (8) consecutive nucleobases selected fromwithin the illustrative antisense compounds are considered to besuitable antisense compounds as well.

[0031] Exemplary preferred antisense compounds include oligonucleotidesequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 8to about 80 nucleobases). Similarly preferred antisense compounds arerepresented by oligonucleotide sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases). Onehaving skill in the art armed with the preferred antisense compoundsillustrated herein will be able, without undue experimentation, toidentify further preferred antisense compounds.

[0032] C. Targets of the Invention

[0033] “Targeting” an antisense compound to a particular nucleic acidmolecule, in the context of this invention, can be a multistep process.The process usually begins with the identification of a target nucleicacid whose function is to be modulated. This target nucleic acid may be,for example, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes Gankyrin.

[0034] The targeting process usually also includes determination of atleast one target region, segment, or site within the target nucleic acidfor the antisense interaction to occur such that the desired effect,e.g., modulation of expression, will result. Within the context of thepresent invention, the term “region” is defined as a portion of thetarget nucleic acid having at least one identifiable structure,function, or characteristic. Within regions of target nucleic acids aresegments. “Segments” are defined as smaller or sub-portions of regionswithin a target nucleic acid. “Sites,” as used in the present invention,are defined as positions within a target nucleic acid.

[0035] Since, as is known in the art, the translation initiation codonis typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding Gankyrin, regardless of the sequence(s)of such codons. It is also known in the art that a translationtermination codon (or “stop codon”) of a gene may have one of threesequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNAsequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

[0036] The terms “start codon region” and “translation initiation codonregion” refer to a portion of such an mRNA or gene that encompasses fromabout 25 to about 50 contiguous nucleotides in either direction (i.e.,5′or 3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense compounds of the presentinvention.

[0037] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

[0038] Other target regions include the 5′ untranslated region (5′UTR),known in the art to refer to the portion of an mRNA in the 5′ directionfrom the translation initiation codon, and thus including nucleotidesbetween the 5′ cap site and the translation initiation codon of an mRNA(or corresponding nucleotides on the gene), and the 3′ untranslatedregion (3′UTR), known in the art to refer to the portion of an mRNA inthe 3′ direction from the translation termination codon, and thusincluding nucleotides between the translation termination codon and 3′end of an mRNA (or corresponding nucleotides on the gene). The 5′ capsite of an mRNA comprises an N7-methylated guanosine residue joined tothe 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′cap region of an mRNA is considered to include the 5′ cap structureitself as well as the first 50 nucleotides adjacent to the cap site. Itis also preferred to target the 5′ cap region.

[0039] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. Targeting splice sites,i.e., intron-exon junctions or exon-intron junctions, may also beparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred target sites. mRNA transcripts producedvia the process of splicing of two (or more) mRNAs from different genesources are known as “fusion transcripts”. It is also known that intronscan be effectively targeted using antisense compounds targeted to, forexample, DNA or pre-mRNA.

[0040] It is also known in the art that alternative RNA transcripts canbe produced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

[0041] Upon excision of one or more exon or intron regions, or portionsthereof during splicing, pre-mRNA variants produce smaller “mRNAvariants”. Consequently, mRNA variants are processed pre-mRNA variantsand each unique pre-mRNA variant must always produce a unique mRNAvariant as a result of splicing. These mRNA variants are also known as“alternative splice variants”. If no splicing of the pre-mRNA variantoccurs then the pre-mRNA variant is identical to the mRNA variant.

[0042] It is also known in the art that variants can be produced throughthe use of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso preferred target nucleic acids.

[0043] The locations on the target nucleic acid to which the preferredantisense compounds hybridize are hereinbelow referred to as “preferredtarget segments.” As used herein the term “preferred target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense compound is targeted. While not wishing to be boundby theory, it is presently believed that these target segments representportions of the target nucleic acid which are accessible forhybridization.

[0044] While the specific sequences of certain preferred target segmentsare set forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional preferred target segments may beidentified by one having ordinary skill.

[0045] Target segments 8-80 nucleobases in length comprising a stretchof at least eight (8) consecutive nucleobases selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

[0046] Target segments can include DNA or RNA sequences that comprise atleast the 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). One having skill in the art armed with the preferredtarget segments illustrated herein will be able, without undueexperimentation, to identify further preferred target segments.

[0047] Once one or more target regions, segments or sites have beenidentified, antisense compounds are chosen which are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, to give the desired effect.

[0048] D. Screening and Target Validation

[0049] In a further embodiment, the “preferred target segments”identified herein may be employed in a screen for additional compoundsthat modulate the expression of Gankyrin. “Modulators” are thosecompounds that decrease or increase the expression of a nucleic acidmolecule encoding Gankyrin and which comprise at least an 8-nucleobaseportion which is complementary to a preferred target segment. Thescreening method comprises the steps of contacting a preferred targetsegment of a nucleic acid molecule encoding Gankyrin with one or morecandidate modulators, and selecting for one or more candidate modulatorswhich decrease or increase the expression of a nucleic acid moleculeencoding Gankyrin. Once it is shown that the candidate modulator ormodulators are capable of modulating (e.g. either decreasing orincreasing) the expression of a nucleic acid molecule encoding Gankyrin,the modulator may then be employed in further investigative studies ofthe function of Gankyrin, or for use as a research, diagnostic, ortherapeutic agent in accordance with the present invention.

[0050] The preferred target segments of the present invention may bealso be combined with their respective complementary antisense compoundsof the present invention to form stabilized double-stranded (duplexed)oligonucleotides.

[0051] Such double stranded oligonucleotide moieties have been shown inthe art to modulate target expression and regulate translation as wellas RNA processsing via an antisense mechanism. Moreover, thedouble-stranded moieties may be subject to chemical modifications (Fireet al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395,854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science,1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998,95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197;Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev.2001, 15, 188-200). For example, such double-stranded moieties have beenshown to inhibit the target by the classical hybridization of antisensestrand of the duplex to the target, thereby triggering enzymaticdegradation of the target (Tijsterman et al., Science, 2002, 295,694-697).

[0052] The compounds of the present invention can also be applied in theareas of drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between Gankyrin and a disease state, phenotype, orcondition. These methods include detecting or modulating Gankyrincomprising contacting a sample, tissue, cell, or organism with thecompounds of the present invention, measuring the nucleic acid orprotein level of Gankyrin and/or a related phenotypic or chemicalendpoint at some time after treatment, and optionally comparing themeasured value to a non-treated sample or sample treated with a furthercompound of the invention. These methods can also be performed inparallel or in combination with other experiments to determine thefunction of unknown genes for the process of target validation or todetermine the validity of a particular gene product as a target fortreatment or prevention of a particular disease, condition, orphenotype.

[0053] E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0054] The compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. Furthermore, antisense oligonucleotides, which are able to inhibitgene expression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

[0055] For use in kits and diagnostics, the compounds of the presentinvention, either alone or in combination with other compounds ortherapeutics, can be used as tools in differential and/or combinatorialanalyses to elucidate expression patterns of a portion or the entirecomplement of genes expressed within cells and tissues.

[0056] As one nonlimiting example, expression patterns within cells ortissues treated with one or more antisense compounds are compared tocontrol cells or tissues not treated with antisense compounds and thepatterns produced are analyzed for differential levels of geneexpression as they pertain, for example, to disease association,signaling pathway, cellular localization, expression level, size,structure or function of the genes examined. These analyses can beperformed on stimulated or unstimulated cells and in the presence orabsence of other compounds which affect expression patterns.

[0057] Examples of methods of gene expression analysis known in the artinclude DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000,480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0058] The compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingGankyrin. For example, oligonucleotides that are shown to hybridize withsuch efficiency and under such conditions as disclosed herein as to beeffective Gankyrin inhibitors will also be effective primers or probesunder conditions favoring gene amplification or detection, respectively.These primers and probes are useful in methods requiring the specificdetection of nucleic acid molecules encoding Gankyrin and in theamplification of said nucleic acid molecules for detection or for use infurther studies of Gankyrin. Hybridization of the antisenseoligonucleotides, particularly the primers and probes, of the inventionwith a nucleic acid encoding Gankyrin can be detected by means known inthe art. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of Gankyrin in a sample may also be prepared.

[0059] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisense compounds havebeen employed as therapeutic moieties in the treatment of disease statesin animals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals, especially humans.

[0060] For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of Gankyrin is treated by administering antisense compoundsin accordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step of administering to the animalin need of treatment, a therapeutically effective amount of a Gankyrininhibitor. The Gankyrin inhibitors of the present invention effectivelyinhibit the activity of the Gankyrin protein or inhibit the expressionof the Gankyrin protein. In one embodiment, the activity or expressionof Gankyrin in an animal is inhibited by about 10%. Preferably, theactivity or expression of Gankyrin in an animal is inhibited by about30%. More preferably, the activity or expression of Gankyrin in ananimal is inhibited by 50% or more.

[0061] For example, the reduction of the expression of Gankyrin may bemeasured in serum, adipose tissue, liver or any other body fluid, tissueor organ of the animal. Preferably, the cells contained within saidfluids, tissues or organs being analyzed contain a nucleic acid moleculeencoding Gankyrin protein and/or the Gankyrin protein itself.

[0062] The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

[0063] F. Modifications

[0064] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

[0065] Modified Internucleoside Linkages (Backbones)

[0066] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0067] Preferred modified oligonucleotide backbones containing aphosphorus atom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0068] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0069] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0070] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0071] Modified Sugar and Internucleoside Linkages-Mimetics

[0072] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage (i.e. the backbone), of the nucleotide unitsare replaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate target nucleic acid. One suchcompound, an oligonucleotide mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0073] Preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0074] Modified Sugars

[0075] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—OCH₂—N(CH₃)₂, also described in examples hereinbelow.

[0076] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0077] A further preferred modification of the sugar includes LockedNucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugarmoiety. The linkage is preferably a methylene (—CH₂—)_(n) group bridgingthe 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0078] Natural and Modified Nucleobases

[0079] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2-(3H)-one),carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are presently preferred base substitutions, even more particularlywhen combined with 2′-O-methoxyethyl sugar modifications.

[0080] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

[0081] Conjugates

[0082] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. These moieties or conjugates caninclude conjugate groups covalently bound to functional groups such asprimary or secondary hydroxyl groups. Conjugate groups of the inventioninclude intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugate groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve uptake,enhance resistance to degradation, and/or strengthen sequence-specifichybridization with the target nucleic acid. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve uptake, distribution, metabolism or excretion of thecompounds of the present invention. Representative conjugate groups aredisclosed in International Patent Application PCT/US92/09196, filed Oct.23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of whichare incorporated herein by reference. Conjugate moieties include but arenot limited to lipid moieties such as a cholesterol moiety, cholic acid,a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999) which is incorporated herein byreference in its entirety.

[0083] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0084] Chimeric Compounds

[0085] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

[0086] The present invention also includes antisense compounds which arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

[0087] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0088] G. Formulations

[0089] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0090] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0091] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0092] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto. For oligonucleotides, preferred examplesof pharmaceutically acceptable salts and their uses are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

[0093] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

[0094] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0095] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0096] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

[0097] Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter. Emulsions may contain additional components in addition to thedispersed phases, and the active drug which may be present as a solutionin either the aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

[0098] Formulations of the present invention include liposomalformulations. As used in the present invention, the term “liposome”means a vesicle composed of amphiphilic lipids arranged in a sphericalbilayer or bilayers. Liposomes are unilamellar or multilamellar vesicleswhich have a membrane formed from a lipophilic material and an aqueousinterior that contains the composition to be delivered. Cationicliposomes are positively charged liposomes which are believed tointeract with negatively charged DNA molecules to form a stable complex.Liposomes that are pH-sensitive or negatively-charged are believed toentrap DNA rather than complex with it. Both cationic and noncationicliposomes have been used to deliver DNA to cells.

[0099] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposomecomprises one or more glycolipids or is derivatized with one or morehydrophilic polymers, such as a polyethylene glycol (PEG) moiety.Liposomes and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

[0100] The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

[0101] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

[0102] One of skill in the art will recognize that formulations areroutinely designed according to their intended use, i.e. route ofadministration.

[0103] Preferred formulations for topical administration include thosein which the oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Preferredlipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidylglycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA).

[0104] For topical or other administration, oligonucleotides of theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively,oligonucleotides may be complexed to lipids, in particular to cationiclipids. Preferred fatty acids and esters, pharmaceutically acceptablesalts thereof, and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999, which is incorporated herein byreference in its entirety.

[0105] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Preferred bile acids/salts and fatty acids and their uses are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety. Also preferred are combinations of penetration enhancers,for example, fatty acids/salts in combination with bile acids/salts. Aparticularly preferred combination is the sodium salt of lauric acid,capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally, in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety. Oral formulations for oligonucleotides and theirpreparation are described in detail in U.S. application Ser. Nos.09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999) and10/071,822, filed Feb. 8, 2002, each of which is incorporated herein byreference in their entirety.

[0106] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0107] Certain embodiments of the invention provide pharmaceuticalcompositions containing one or more oligomeric compounds and one or moreother chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to cancer chemotherapeutic drugs such as daunorubicin,daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin,esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Antiinflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

[0108] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Alternatively,compositions of the invention may contain two or more antisensecompounds targeted to different regions of the same nucleic acid target.Numerous examples of antisense compounds are known in the art. Two ormore combined compounds may be used together or sequentially.

[0109] H. Dosing

[0110] The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 ugto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0111] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Synthesis of Nucleoside Phosphoramidites

[0112] The following compounds, including amidites and theirintermediates were prepared as described in U.S. Pat. No. 6,426,220 andpublished PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediatefor 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidineintermediate for 5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2 Oligonucleotide and Oligonucleoside Synthesis

[0113] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0114] Oligonucleotides: Unsubstituted and substituted phosphodiester(P═O) oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

[0115] Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

[0116] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0117] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0118] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0119] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0120] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0121] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0122] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

[0123] Oligonucleosides: Methylenemethylimino linked oligonucleosides,also identified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0124] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0125] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3 RNA Synthesis

[0126] In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

[0127] Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

[0128] RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

[0129] Following synthesis, the methyl protecting groups on thephosphates are cleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

[0130] The 2′-orthoester groups are the last protecting groups to beremoved. The ethylene glycol monoacetate orthoester protecting groupdeveloped by Dharmacon Research, Inc. (Lafayette, Colo.), is one exampleof a useful orthoester protecting group which, has the followingimportant properties. It is stable to the conditions of nucleosidephosphoramidite synthesis and oligonucleotide synthesis. However, afteroligonucleotide synthesis the oligonucleotide is treated withmethylamine which not only cleaves the oligonucleotide from the solidsupport but also removes the acetyl groups from the orthoesters. Theresulting 2-ethylhydroxyl substituents on the orthoester are lesselectron withdrawing than the acetylated precursor. As a result, themodified orthoester becomes more labile to acid-catalyzed hydrolysis.Specifically, the rate of cleavage is approximately 10 times fasterafter the acetyl groups are removed. Therefore, this orthoesterpossesses sufficient stability in order to be compatible witholigonucleotide synthesis and yet, when subsequently modified, permitsdeprotection to be carried out under relatively mild aqueous conditionscompatible with the final RNA oligonucleotide product.

[0131] Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

[0132] RNA antisense compounds (RNA oligonucleotides) of the presentinvention can be synthesized by the methods herein or purchased fromDharmacon Research, Inc (Lafayette, Colo.). Once synthesized,complementary RNA antisense compounds can then be annealed by methodsknown in the art to form double stranded (duplexed) antisense compounds.For example, duplexes can be formed by combining 30 μl of each of thecomplementary strands of RNA oligonucleotides (50 um RNA oligonucleotidesolution) and 15 μl of 5× annealing buffer (100 mM potassium acetate, 30mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisensecompounds can be used in kits, assays, screens, or other methods toinvestigate the role of a target nucleic acid.

Example 4 Synthesis of Chimeric Oligonucleotides

[0133] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0134] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[0135] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxyPhosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0136] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxyphosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0137] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 Design and Screening of Duplexed Antisense Compounds TargetingGankyrin

[0138] In accordance with the present invention, a series of nucleicacid duplexes comprising the antisense compounds of the presentinvention and their complements can be designed to target Gankyrin. Thenucleobase sequence of the antisense strand of the duplex comprises atleast a portion of an oligonucleotide in Table 1. The ends of thestrands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini.

[0139] For example, a duplex comprising an antisense strand having thesequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang ofdeoxythymidine(dT) would have the following structure:  cgagaggcggacgggaccgTT Antisense Strand   |||||||||||||||||||TTgctctccgcctgccctggc Complement

[0140] RNA strands of the duplex can be synthesized by methods disclosedherein or purchased from Dharmacon Research Inc., (Lafayette, Colo.).Once synthesized, the complementary strands are annealed. The singlestrands are aliquoted and diluted to a concentration of 50 um. Oncediluted, 30 uL of each strand is combined with 15 uL of a 5× solution ofannealing buffer. The final concentration of said buffer is 100 mMpotassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate.The final volume is 75 uL. This solution is incubated for 1 minute at90° C. and then centrifuged for 15 seconds. The tube is allowed to sitfor 1 hour at 37° C. at which time the dsRNA duplexes are used inexperimentation. The final concentration of the dsRNA duplex is 20 uM.This solution can be stored frozen (−20° C.) and freeze-thawed up to 5times.

[0141] Once prepared, the duplexed antisense compounds are evaluated fortheir ability to modulate Gankyrin expression.

[0142] When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at afinal concentration of 200 nM. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

Example 6 Oligonucleotide Isolation

[0143] After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

[0144] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a 96-well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

[0145] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96-Well Plate Format

[0146] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

[0147] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,ribonuclease protection assays, or RT-PCR.

[0148] T-24 Cells:

[0149] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #353872) at a density of7000 cells/well for use in RT-PCR analysis.

[0150] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0151] A549 Cells:

[0152] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

[0153] NHDF Cells:

[0154] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0155] HEK Cells:

[0156] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.). HEKs were routinelymaintained in Keratinocyte Growth Medium (Clonetics Corporation,Walkersville, Md.) formulated as recommended by the supplier. Cells wereroutinely maintained for up to 10 passages as recommended by thesupplier.

[0157] Treatment with Antisense Compounds:

[0158] When cells reached 65-75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.Cells are treated and data are obtained in triplicate. After 4-7 hoursof treatment at 37° C., the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

[0159] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of cH-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of Gankyrin Expression

[0160] Antisense modulation of Gankyrin expression can be assayed in avariety of ways known in the art. For example, Gankyrin mRNA levels canbe quantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis ofthe present invention is the use of total cellular RNA as described inother examples herein. Methods of RNA isolation are well known in theart. Northern blot analysis is also routine in the art. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

[0161] Protein levels of Gankyrin can be quantitated in a variety ofways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed toGankyrin can be identified and obtained from a variety of sources, suchas the MSRS catalog of antibodies (Aerie Corporation, Birmingham,Mich.), or can be prepared via conventional monoclonal or polyclonalantibody generation methods well known in the art.

Example 11 Design of phenotypic Assays and in Vivo Studies for the Useof Gankyrin Inhibitors

[0162] Phenotypic Assays

[0163] Once Gankyrin inhibitors have been identified by the methodsdisclosed herein, the compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of Gankyrin in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

[0164] In one non-limiting example, cells determined to be appropriatefor a particular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated withGankyrin inhibitors identified from the in vitro studies as well ascontrol compounds at optimal concentrations which are determined by themethods described above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

[0165] Phenotypic endpoints include changes in cell morphology over timeor treatment dose as well as changes in levels of cellular componentssuch as proteins, lipids, nucleic acids, hormones, saccharides ormetals. Measurements of cellular status which include pH, stage of thecell cycle, intake or excretion of biological indicators by the cell,are also endpoints of interest.

[0166] Analysis of the geneotype of the cell (measurement of theexpression of one or more of the genes of the cell) after treatment isalso used as an indicator of the efficacy or potency of the Gankyrininhibitors. Hallmark genes, or those genes suspected to be associatedwith a specific disease state, condition, or phenotype, are measured inboth treated and untreated cells.

[0167] In vivo Studies

[0168] The individual subjects of the in vivo studies described hereinare warm-blooded vertebrate animals, which includes humans.

[0169] The clinical trial is subjected to rigorous controls to ensurethat individuals are not unnecessarily put at risk and that they arefully informed about their role in the study. To account for thepsychological effects of receiving treatments, volunteers are randomlygiven placebo or Gankyrin inhibitor. Furthermore, to prevent the doctorsfrom being biased in treatments, they are not informed as to whether themedication they are administering is a Gankyrin inhibitor or a placebo.Using this randomization approach, each volunteer has the same chance ofbeing given either the new treatment or the placebo.

[0170] Volunteers receive either the Gankyrin inhibitor or placebo foreight week period with biological parameters associated with theindicated disease state or condition being measured at the beginning(baseline measurements before any treatment), end (after the finaltreatment), and at regular intervals during the study period. Suchmeasurements include the levels of nucleic acid molecules encodingGankyrin or Gankyrin protein levels in body fluids, tissues or organscompared to pre-treatment levels. Other measurements include, but arenot limited to, indices of the disease state or condition being treated,body weight, blood pressure, serum titers of pharmacologic indicators ofdisease or toxicity as well as ADME (absorption, distribution,metabolism and excretion) measurements.

[0171] Information recorded for each patient includes age (years),gender, height (cm), family history of disease state or condition(yes/no), motivation rating (some/moderate/great) and number and type ofprevious treatment regimens for the indicated disease or condition.

[0172] Volunteers taking part in this study are healthy adults (age 18to 65 years) and roughly an equal number of males and femalesparticipate in the study. Volunteers with certain characteristics areequally distributed for placebo and Gankyrin inhibitor treatment. Ingeneral, the volunteers treated with placebo have little or no responseto treatment, whereas the volunteers treated with the Gankyrin inhibitorshow positive trends in their disease state or condition index at theconclusion of the study.

Example 12 RNA Isolation

[0173] Poly(A)+ mRNA isolation

[0174] Poly(A)+ mRNA was isolated according to Miura et al., (Clin.Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolationare routine in the art. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA,0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was addedto each well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine, Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

[0175] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

[0176] Total RNA Isolation

[0177] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 150 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 150 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and incubated for 15 minutes and the vacuum was again applied for1 minute. An additional 500 μL of Buffer RW1 was added to each well ofthe RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL ofBuffer RPE was then added to each well of the RNEASY 96™ plate and thevacuum applied for a period of 90 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 3 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 140 μL of RNAse free water into eachwell, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0178] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia, Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-time Quantitative PCR Analysis of Gankyrin mRNA Levels

[0179] Quantitation of Gankyrin mRNA levels was accomplished byreal-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. This is a closed-tube,non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR in which amplification productsare quantitated after the PCR is completed, products in real-timequantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 5′ end of the probe and a quencherdye (e.g., TAMRA, obtained from either PE-Applied Biosystems, FosterCity, Calif., Operon Technologies Inc., Alameda, Calif. or IntegratedDNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ Sequence Detection System. In each assay, aseries of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

[0180] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0181] PCR reagents were obtained from Invitrogen Corporation,(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μLPCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 MM each ofdATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-wellplates containing 30 μL total RNA solution (20-200 ng). The RT reactionwas carried out by incubation for 30 minutes at 48° C. Following a 10minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles ofa two-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0182] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methodsof RNA quantification by RiboGreen™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

[0183] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 30 μL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at485 nm and emission at 530 nm.

[0184] Probes and primers to human Gankyrin were designed to hybridizeto a human Gankyrin sequence, using published sequence information(GenBank accession number NT_(—)011765.5, incorporated herein as SEQ IDNO: 4). For human Gankyrin the PCR primers were: forward primer:TGCTGGCCGGGATGAG (SEQ ID NO: 5) reverse primer: CCATTTTGATTGACAGCATTCAC(SEQ ID NO: 6) and the PCR probe was:FAM-TGTAAAAGCCCTTCTGGGAAAAGGTGCTC-TAMRA (SEQ ID NO: 7) where FAM is thefluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCRprimers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) reverseprimer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is thefluorescent reporter dye and TAMRA is the quencher dye.

Example 14 Northern Blot Analysis of Gankyrin mRNA Levels

[0185] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0186] To detect human Gankyrin, a human Gankyrin specific probe wasprepared by PCR using the forward primer TGCTGGCCGGGATGAG (SEQ ID NO: 5)and the reverse primer CCATTTTGATTGACAGCATTCAC (SEQ ID NO: 6). Tonormalize for variations in loading and transfer efficiency membraneswere stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0187] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense Inhibition of Human Gankyrin Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0188] In accordance with the present invention, a series of antisensecompounds were designed to target different regions of the humanGankyrin RNA, using published sequences (nucleotides 658700 to 669109 ofthe sequence with GenBank accession number NT_(—)011765.5, incorporatedherein as SEQ ID NO: 4, GenBank accession number NM_(—)002814.1,incorporated herein as SEQ ID NO: 11, GenBank accession numberAA326291.1, incorporated herein as SEQ ID NO: 12, and GenBank accessionnumber BG831521.1, incorporated herein as SEQ ID NO: 13). The compoundsare shown in Table 1. “Target site” indicates the first (5′-most)nucleotide number on the particular target sequence to which thecompound binds. All compounds in Table 1 are chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings”. The wings arecomposed of 2′-methoxyethyl (2′MOE) nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. Thecompounds were analyzed for their effect on human Gankyrin mRNA levelsby quantitative real-time PCR as described in other examples herein.Data are averages from three experiments in which T-24 cells weretreated with the antisense oligonucleotides of the present invention. Ifpresent, “N.D.” indicates “no data”. TABLE 1 Inhibition of humanGankyrin mRNA levels by chimeric phosphorothioate oligonucleotideshaving 2′-MOE wings and a deoxy gap TARGET SEQ ID TARGET % SEQ ID ISIS #REGION NO SITE SEQUENCE INHIB NO 170972 exon 4 8115 ccattctcttgagtattaaa59 14 170973 3′UTR 4 8644 ctaaaatatgtaaatatccc 0 15 170974 exon 4 1631tcccgctgtaggccaggttg 52 16 170975 3′UTR 4 8750 acattttatttcagagtaaa 2 17170976 3′UTR 4 8528 actgtatggaggaacattac 36 18 170977 3′UTR 4 8677tagagcacaccatccaactt 19 19 170978 5′UTR 4 1525 gctccggctacgccagtcaa 0 20170979 exon 4 5374 ctttgtagtacagaaggata 45 21 170980 3′UTR 4 8468tgctattggagctgttctgt 63 22 170981 Coding 11 298 acctgcatcgtctttatcat 3323 170982 exon 4 1621 ggccaggttgcagaccatta 72 24 170983 3′UTR 4 8320tattcgatgcctggaaaaca 50 25 170984 exon 4 5085 tcacttgagcaccttttccc 55 26170985 Coding 11 444 gcgatctcatgcctgttttt 62 27 170986 exon 4 8109tcttgagtattaaacccagg 74 28 170987 exon 4 4306 ttcaacaatttctgtatgtc 25 29170988 exon 4 1666 atcggccagaatactctcct 70 30 170989 exon 4 5088cattcacttgagcacctttt 43 31 170990 exon 4 5138 gtttttcgaagctgcataat 23 32170991 3′UTR 4 8211 atgatgtcttgtgcacaaat 67 33 170992 3′UTR 4 8260tcaacatgtttataagactt 61 34 170993 3′UTR 4 8784 taggtactttaaaacttcct 5535 170994 Start 4 1580 tttcgctgtcccagcaacta 51 36 Codon 170995 3′UTR 48326 aacagttattcgatgcctgg 85 37 170996 exon 4 4279 gcatgcccagtgcaatgcag37 38 170997 3′UTR 4 8765 ttttaagaaaaccatacatt 6 39 170998 3′UTR 4 8208atgtcttgtgcacaaataca 83 40 170999 exon 4 1676 ccagggatttatcggccaga 80 41171000 exon 4 8097 aacccaggccacctttggcc 60 42 171001 exon 4 4282tgagcatgcccagtgcaatg 48 43 171002 3′UTR 4 8227 acttcatcattcatagatga 4144 171003 exon: 4 5345 ttcaagttacccttggctgc 75 45 intron junction 171004exon: 4 5348 atcttcaagttacccttggc 48 46 intron junction 171005 5′UTR 41506 aaacagccgttagagcttca 0 47 171006 exon 4 5383 ttgtggatgctttgtagtac36 48 171007 exon 4 4277 atgcccagtgcaatgcagtt 54 49 171008 3′UTR 4 8251ttataagactttgaaggtga 40 50 224117 Start 4 1591 acacccctccatttcgctgt 8851 Codon 224118 Start 4 1601 ggttagacacacacccctcc 77 52 Codon 224119Coding 11 203 ttctgctgtcctggtcagtt 51 53 224120 exon 4 4330tggcactccaagttgcaaca 70 54 224121 exon 4 5029 gccgcaatatgaagaggaga 69 55224122 exon 4 5113 ggagtacagccattttgatt 69 56 224123 exon 4 5272cgccttccagtaacatgaca 28 57 224124 exon 4 5298 atggtccttagcatctggat 76 58224125 exon 4 5317 gcattgctgtagcctcataa 81 59 224126 exon 4 5334cttggctgctgcccggtgca 74 60 224127 Coding 11 617 aggctaagtgtagaggagtg 2061 224128 exon 4 8000 cactctctcctcatcacagg 67 62 224129 exon 4 8022acaccagcagttttgcttct 65 63 224130 exon 4 8044 atgtaaatacttgctccttg 70 64224131 Stop 4 8135 ccaagctgtttaaccttcca 79 65 Codon 224132 3′UTR 4 8146taagaataaatccaagctgt 81 66 224133 3′UTR 4 8420 gggtgctgaagactcacaac 6567 224134 3′UTR 4 8448 ttcagagagggatataaggt 70 68 224135 3′UTR 4 8486gcagaacaactagcttgttg 72 69 224136 3′UTR 4 8540 taggatgttttaactgtatg 7770 224137 3′UTR 4 8660 cttaaaatgtggtccactaa 79 71 224138 3′UTR 4 8822caagcttgacattcttttca 63 72 224139 3′UTR 4 8870 gtttctgaaatacaaatcaa 5173 224140 exon: 4 1703 gttgctttacctggtcagtt 65 74 intron junction 224141intron 4 2180 agcaccatgtaaacttctct 72 75 224142 intron 4 2429tttgtgggcttactaaaggc 46 76 224143 intron 4 2822 gcagttggtttgtttaaatc 5277 224144 intron: 4 5010 accaacctgcctataaaaga 14 78 exon junction 224145exon: 4 5424 tgtcacttacagaggagtgt 60 79 intron junction 224146 intron 47022 taatactttttaaaagttgg 8 80 224147 intron: 4 7982ggctaagtgtctgaaatcag 55 81 exon junction 224148 exon: 12 110accaacctgcctggtcagtt 28 82 exon junction 224149 exon: 13 449ggctaagtgtccttggctgc 24 83 exon junction

[0189] As shown in Table 1, SEQ ID NOs 14, 16, 21, 22, 24, 25, 26, 27,28, 30, 31, 33, 34, 35, 36, 37, 40, 41, 42, 43, 44, 45, 46, 49, 50, 51,52, 53, 54, 55, 56, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 79 demonstrated at least 40% inhibition of humanGankyrin expansion in this assay and are therefore preferred. Morepreferred are SEQ ID NOs 41, 37 and 40. The target regions to whichthese preferred sequences are complementary are herein referred to as“preferred target segments” and are therefore preferred for targeting bycompounds of the present invention. These preferred target segments areshown in Table 2. The sequences represent the reverse complement of thepreferred antisense compounds shown in Table 1. “Target site” indicatesthe first (5′-most) nucleotide number on the particular target nucleicacid to which the oligonucleotide binds. Also shown in Table 2 is thespecies in which each of the preferred target segments was found. TABLE2 Sequence and position of preferred target segments identified inGankyrin. TARGET SITE SEQ ID TARGET REV COMP SEQ ID ID NO SITE SEQUENCEOF SEQ ID ACTIVE IN NO 86100 4 8115 tttaatactcaagagaatgg 14 H. sapiens84 86102 4 1631 caacctggcctacagcggga 16 H. sapiens 85 86107 4 5374tatccttctgtactacaaag 21 H. sapiens 86 86108 4 8468 acagaacagctccaatagca22 H. sapiens 87 86110 4 1621 taatggtctgcaacctggcc 24 H. sapiens 8886111 4 8320 tgttttccaggcatcgaata 25 H. sapiens 89 86112 4 5085gggaaaaggtgctcaagtga 26 H. sapiens 90 86113 11 444 aaaaacaggcatgagatcgc27 H. sapiens 91 86114 4 8109 cctgggtttaatactcaaga 28 H. sapiens 9286116 4 1666 aggagagtattctggccgat 30 H. sapiens 93 86117 4 5088aaaaggtgctcaagtgaatg 31 H. sapiens 94 86119 4 8211 atttgtgcacaagacatcat33 H. sapiens 95 86120 4 8260 aagtcttataaacatgttga 34 H. sapiens 9686121 4 8784 aggaagttttaaagtaccta 35 H. sapiens 97 86122 4 1580tagttgctgggacagcgaaa 36 H. sapiens 98 86123 4 8326 ccaggcatcgaataactgtt37 H. sapiens 99 86126 4 8208 tgtatttgtgcacaagacat 40 H. sapiens 10086127 4 1676 tctggccgataaatccctgg 41 H. sapiens 101 86128 4 8097ggccaaaggtggcctgggtt 42 H. sapiens 102 86129 4 4282 cattgcactgggcatgctca43 H. sapiens 103 86130 4 8227 tcatctatgaatgatgaagt 44 H. sapiens 10486131 4 5345 gcagccaagggtaacttgaa 45 H. sapiens 105 86132 4 5348gccaagggtaacttgaagat 46 H. sapiens 106 86135 4 4277 aactgcattgcactgggcat49 H. sapiens 107 86136 4 8251 tcaccttcaaagtcttataa 50 H. sapiens 108140771 4 1591 acaqcgaaatggaggggtgt 51 H. sapiens 109 140772 4 1601ggaggggtgtgtgtctaacc 52 H. sapiens 110 140773 11 203aactgaccaggacagcagaa 53 H. sapiens 111 140774 4 4330tgttgcaacttggagtgcca 54 H. sapiens 112 140775 4 5029tctcctcttcatattgcggc 55 H. sapiens 113 140776 4 5113aatcaaaatggctgtactcc 56 H. sapiens 114 140778 4 5298atccagatgctaaggaccat 58 H. sapiens 115 140779 4 5317ttatgaggctacagcaatgc 59 H. sapiens 116 140780 4 5334tgcaccgggcagcagccaag 60 H. sapiens 117 140782 4 8000cctgtgatgaggagagagtg 62 H. sapiens 118 140783 4 8022agaagcaaaactgctggtgt 63 H. sapiens 119 140784 4 8044caaggagcaagtatttacat 64 H. sapiens 120 140785 4 8135tggaaggttaaacagcttgg 65 H. sapiens 121 140786 4 8146acagcttggatttattctta 66 H. sapiens 122 140787 4 8420gttgtgagtcttcagcaccc 67 H. sapiens 123 140788 4 8448accttatatccctctctgaa 68 H. sapiens 124 140789 4 8486caacaagctagttgttctgc 69 H. sapiens 125 140790 4 8540catacagttaaaacatccta 70 H. sapiens 126 140791 4 8660ttagtggaccacattttaag 71 H. sapiens 127 140792 4 8822tgaaaagaatgtcaagcttg 72 H. sapiens 128 140793 4 8870ttgatttgtatttcagaaac 73 H. sapiens 129 140794 4 1703aactgaccaggtaaagcaac 74 H. sapiens 130 140795 4 2180agagaagtttacatggtgct 75 H. sapiens 131 140796 4 2429gcctttagtaagcccacaaa 76 H. sapiens 132 140797 4 2822gatttaaacaaaccaactgc 77 H. sapiens 133 140799 4 5424acactcctctgtaagtgaca 79 H. sapiens 134 140801 4 7982ctgatttcagacacttagcc 81 H. sapiens 135

[0190] As these “preferred target segments” have been found byexperimentation to be open to, and accessible for, hybridization withthe antisense compounds of the present invention, one of skill in theart will recognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of Gankyrin.

[0191] According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other short oligomeric compounds which hybridize toat least a portion of the target nucleic acid.

Example 16 Western Blot Analysis of Gankyrin Protein Levels

[0192] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to Gankyrin is used,with a radiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale, Calif.).

1 135 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence AntisenseOligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial SequenceAntisense Oligonucleotide 3 atgcattctg cccccaagga 20 4 10410 DNA H.sapiens 4 aaaaatcatt taattatgca cttgaaatca gtgaatttta tgatatgtaaaatgtgcctc 60 attttgagtg agtgttatac aggaactcta ctaaccttgc aacttttctataaatctaca 120 actattctga aatttttgaa aaattaaata aaaataaaaa taaaaaagtatatgtttatg 180 aattgatatc agtatttggc agtacctatc tatgtaaaat gacattttcaaagattaaat 240 aggtaaaaat ttcctaacag atcagcatta acagatgaac acttaaaatcaattttgaca 300 atagggaaca ctaattctga gccccaatta agcaaaatat tatcccctaaaaagaattct 360 attcttctca ttggtagtct tcctgtatta caaaaaaagt actcaattactattatattt 420 tgaatttcat caataaaaat ttgttgaaat ttgttttctc ttttgtcataaaagtaccaa 480 caaaatattc ttgattttgc cttttggcct acaaagccta aaatatttactatctggctc 540 tttacagaaa gtttcccaac tcctgttcta cactataaca ggataaacataaacatcatg 600 agtctctttc ttgaatgtat tcttatcgtt acctagttgt ggcataatgtcctctggata 660 tgccctataa aggatcagtc ttgcattgct tctgctatgg cactatttgggatttctata 720 aaaagaaaca ctattagctt aatagttgta gcaaagcctt gacaacatagttaatatcgt 780 gatattaaaa tgccaacacc acagcccggt atccttaaga tgaccctcatcaagctgtga 840 tgtcaccagg ctatggactc accgaggtct cttagatatc aggtggctcccacggcatca 900 caccctgcct ttggcatcac cctggcacat cacctcagta cggtgccatatgctatcact 960 gctgtggtag aaccctgtcc tgatgccaca ccgccgagat gtcagagtaattaaccccca 1020 cttctttcca ctccctaggc ggtggcaggg agggggaggg tagccacagtcaccggttcg 1080 agctcagtag gtagtactca gctccaaccc ttgtcccttc cgcctgccactgcaggtcag 1140 gaaccccggg ggacaggacc tttaggctct cataactctt gccctgcccccgaacctgtt 1200 tcatatggcc gactcaacga cccgccagcg actacccacc ccggctctttcacacggttc 1260 cactcaaagc cgctccccgt acctgactcc attctccagc tgtcatcctgggccaattcc 1320 accggcaact tgactacgga cggacggtcg ctaggatccc tgggacttgtagttctgcac 1380 tgctaggggc caagtctgtg agggcagcaa aggcgcctcc ctgcccggaactgctctcaa 1440 actgcaactc ccagaggcgc cgcgcgacgg gaaaagaaaa gggaacgaggaaggccggtg 1500 attggtgaag ctctaacggc tgttttgact ggcgtagccg gagccggcgacgtgaggcgg 1560 gcgttgctcg cgcgacaagt agttgctggg acagcgaaat ggaggggtgtgtgtctaacc 1620 taatggtctg caacctggcc tacagcggga agctggaaga gttgaaggagagtattctgg 1680 ccgataaatc cctggctact agaactgacc aggtaaagca acgctaggtctctccgcagt 1740 cggcgacgtc ggcgaagccc tacgtggctt tcggggcccg gccctagcggaggccccgtt 1800 tctgagagaa gcagaacccc gcccccgccg ccccaagcag tccccctgggtcacctcggg 1860 actcgcgata ggcctgcggg agctagcccc acagacctca gatattttcccgttggcccg 1920 gagctccgct ccctgatttt tggatagttc tagaacgtct accgcatttctcagaaactc 1980 ccaatcagtg aaaaggaagg ctttttatat ggatttattt acctttactagtaactgtat 2040 tcagtaacgc atgtgaaatt gaacgcccct tccgtgccag gcagtttgctaggagctggg 2100 gatacggtgg agtggaagat gcagacaata aaataagcac aaaaacatgtccgattacaa 2160 attgtggtaa atactcggca gagaagttta catggtgctt tcagaacacgttaataggag 2220 gatctgatgc tgaatatgac tgtcgttaat tggaaaagtt tttgcaaagaagggaaggtt 2280 agttagactt tgaagcatga gtgggatttc catgggggaa tgagtggacattctaagcag 2340 aggtagacag aagtttagaa gtgtttcttt ctgccctcct gctcacaggactctcgtgct 2400 ttcatcctca gtttctagga ttcatggtgc ctttagtaag cccacaaatatttatagatg 2460 gccttgtgtt tcgcagacag tgtcgtaagc actagtgaga ctgtcccatacttacagatc 2520 tccattttgt gtcttttggg agagaattag aaatcaagag actgttttctcttttctgga 2580 actttccttt attactcaac ctgaaaatgg ccttatcttt acctggtttacttcattatg 2640 gtctggtgat atttaaggat ttaaatttaa atgtaatact ttatattaagatttaatact 2700 ccattggaaa tttaaccatt cttcagtcaa gtcagtggct taaaaagggaaaggaactat 2760 tcagcatcaa gagacgtaaa aaacatgaag aaatgcaatt tatggatctcgtctggttcc 2820 tgatttaaac aaaccaactg caaaaagaca tttgggagac agttgggggcaatctgaata 2880 cagaccggat attagacatt ctaaaggagt tagtgttaat tttagtaagtgtaataatgg 2940 tctcatagtt atataagaaa tacctttatt ttttacatac tgaaagagaagggtgaaaag 3000 tcatgatatt taggatttgt ttttggaaag ctcagcaagg aaaaaactagttaaagcaaa 3060 tagggcaaga tgtttgttgt taaatctaag cagtaggcct gtgggtttctattctattcc 3120 ctctattttt gtgtttgaaa tttttttgta atagagttta aaataccattttctattctg 3180 tggggctaag aagtatagca gtcactgcaa atagggattc cctttgacaaataatgacta 3240 ctatagcttt tttaattttt atttattttt atttatttat tttttgaaacagagtttcgc 3300 tcttgttgcc caggctggag tgcagtggtg ccgtatcacc tcactgcaacctctgcctcc 3360 cgggttcaag cgattcccct gcctcagcct cccaagtagc tgggattacaggcgcctgcc 3420 accacgcctg gctaactttt gtatttttag tagagacgga gtttcaccatgttgccaggc 3480 tggtctagaa ctcctgacct cgggtgatct gcccacctca gcctcccaaagtgctgggat 3540 tataggcgtg aaccaccgca cccggccata gcttattatt ttttataaaaagtaattttc 3600 ttgttttctg tgtgttgtgt tttctaaatt atatttctaa tttatttatgattttaaatt 3660 ctagataatt tcaactagtt taatgaacat ttcttccccc attagattcatgtgttcctt 3720 ctacagcatc taacttgagg tcagtgtggt agtagagatt tcagagtcagaacttcactg 3780 acagttgtgc cccaacttca ccatttagta tccatatgac cgtccacaagttgcttaccc 3840 tgaggagttt tagtttcctc atctgaaaaa tggagataat aagcactgtgtagaatggta 3900 ggaattaaat gatactatac atgtaaaact cccagcacag tgcctgacataaaagtacgt 3960 aaataatgta atagtaaaac aaccaaagat gtatttctgt caaagcattgaaatcagttt 4020 cttgctctac tactttcctt gtcgtattct gcccagaatt acgtatgttcatcaatgtca 4080 ttctccccat gttacttaaa ccttcatctg tcttatacca gtaagtaatatagtttaagg 4140 ttacctacct gaggatccag tgctctgcat ttcgtatttt caaactatagatttgccagc 4200 accagtattt tttttcttgc ttttttgcct tctatttcaa gaatgtttctattactgctt 4260 ctctgtagga cagcagaact gcattgcact gggcatgctc agctggacatacagaaattg 4320 ttgaattttt gttgcaactt ggagtgccag tgaatgataa agacgatgtgagtactttga 4380 taaagttgag ttgataaatt cccagttatc tttgtactcc agactggtgtttactgaagc 4440 ctgtgcatta agcttataaa gaaaattgta acctttatga ctggaaagttacatcatggc 4500 ccttgtgatc aagcatgaga ctcatggttg cattatttta caatgttatgtttgccactc 4560 atactctgtg atttaaggta aacatacagg agaggctgag gtaggaggattgctccagcc 4620 tagtagtttg aggctgcagt gagccattgt actctatcct gggtgacagactgagacctt 4680 gtctctaaaa ataaaaaaac aaaaataaac atacagatca gggcttgttatggtgaaccc 4740 taaaagacca taaactcttt ctattatgtt ggattttttg ttttttggtgaacatggcct 4800 gaataatgag gacctaaata acttttttgt tacataaaga gatttattctaatgatatgg 4860 ttatattagg attagatttg tgaagagtaa gaatggaacc ttttttgtaggcaaatattt 4920 ttttgccttt gtaaagctcc ttgctaaccc ttcctaaatg ttagcactaatccttgtgcc 4980 tttttcccct ggtatttcta ctacctactt cttttatagg caggttggtctcctcttcat 5040 attgcggctt ctgctggccg ggatgagatt gtaaaagccc ttctgggaaaaggtgctcaa 5100 gtgaatgctg tcaatcaaaa tggctgtact cccttacatt atgcagcttcgaaaaacagg 5160 catgaggtat ggttccatcc taggctactt ctgttgagtt ctaaacgtgtatacaaacac 5220 agatagattt ctgcattgtt ttcttctttc ttctccatct ttccagatcgctgtcatgtt 5280 actggaaggc ggggctaatc cagatgctaa ggaccattat gaggctacagcaatgcaccg 5340 ggcagcagcc aagggtaact tgaagatgat tcatatcctt ctgtactacaaagcatccac 5400 aaacatccaa gacactgagg gtaacactcc tctgtaagtg acaagtagcagtcatttgta 5460 ttctacctga cattagcctt cttgcaataa ttcttataca tcatttctttttttattctg 5520 cttataaatg caatacgtgt taatggcaaa aatgttaaat tgcacaaaatataaagataa 5580 aaattgccta ttatcttgcc actcagaaat aactactgat cacattcttgtgtatttcct 5640 cctggtcttt tatattcaaa tgtatatatt ttatacatac atattttttagcaaaattgg 5700 ggtcatactg tgtatgtagt tttagctctt gttcttttca cctcatagtataagcatata 5760 ttaaatatgt ttaaaacctt tttaatgggt atataaaatt ctgtatagatgtgccataat 5820 ttaatcattc ctctattact gtatatttaa acttttatga gtttttcactattataaata 5880 atggtacatt gagccaggca taatggctca tgtctgtaat cccagcactttgtaaagcca 5940 aggcgggagg atcacttgag gccaggagtt caagaccagc ctgggcaacacagcaagacc 6000 ccccgtctct agaaaaaatt aaaaacttag ctaggcatgg tggcatgcacctgtagtact 6060 aactactcgg gaggttgagg tgggaggatt gcttgaaccc aggagctccaggttacagtg 6120 agctgtgatc acaccactgc actccagcct ggggaacaga gcaagaccttgtctctaaaa 6180 tagttgacca actttgtaca gaaaactttt ggccacattt ctgatgattaccttcttata 6240 gactcctaga agtggaattg ctgcgtcaac aaatacacac attttaagacttgctatatg 6300 ttgccaaatt gcttccagag gagttgtatc attttaccct cccaacagcagtgtaagagt 6360 gcctattacc ccctcaccaa cactaaatta tccttttgtt atgtgaaaatgatatttcat 6420 agaggtaatt tatattttta ttatcagtga atttgaactt tttttaatgtccataggtaa 6480 ttacatttct tatctaattg taactttttg tgtccctttc tctgtttccttttggggaat 6540 taatttattt tcactgggtt ttaagtgctc ttgatttggc atggtaaaatattaattttc 6600 tgcaatgtta gttgaaaata tttttcctag tttatctttt cattttgttcataatttctt 6660 ttacataaga gaagttttaa aatttctacg tagtcaagcc cattcatctttcctcatgac 6720 tagaaagctc ttccttaccc tgaaattagt taaatattca cccgtcttttattataggtt 6780 attttaacgt ttcattggtt ttgattgagg gttagtgtta ttgttttgcatttaactctt 6840 aaaatccatc tgtaattgaa ttatgtgtat gatctgaagc tggcctttaccttgatattt 6900 ttccacggta cttggccact tttcttctgt gttgaatggt gactttttcttgaaccagaa 6960 gttctgtgag tcttcttttt agtccagagt gaggttcagc cttttccaatttgactttaa 7020 gccaactttt aaaaagtatt attttcttaa ctcttagttt ttaaaatctttcctgcattc 7080 taagagaccc tgtttagaaa gacctgtatg ccctacctct tttctccataaatagaaagg 7140 caggatggcc tagaggaaat ctaggagtca gacaagatag agtactgggacgaacatagg 7200 atttatgaat agctagatga tgttcaccca attcagactc taccacttactaacaatatt 7260 aaatacttgt attaaacaat ttataaaagt tacttaatct tccttgaatcttaatttcct 7320 catctgtaat ataagggtaa taattccttc ctcactagat tattgtgaggtttaagtaaa 7380 gcaacatgta aagtgcctcc taataccctc ctggcaaact cttaagtgcatggcaaaagt 7440 tgttaccctc ttccactcag tctactgcca agacatgtca gcataggtaggaactttaac 7500 ctctgtcacc atttcaactg taaaatgatg ataataatac tttgctaacttataagatag 7560 atatatggac taaatgagat aaaatctttt aaactgcttt ttcaaactttaagctgcacg 7620 aattactatt aacagtttat gaactgaggc ttttctaaat tctaaccctgaattattaca 7680 tgtagtgttt gaggtaaaca actacttatc tctgtaaaat gagaaagttgttccttaggc 7740 tagcatttag atcccttctt ctattgtaag tagaaccaga attatgatttacgatctggt 7800 attacaaggc tttttttgta gaattttaac ttacagtctc cctatgcaaactgatgtttc 7860 tttcctgatt aaataacttt ccaatgttac catttcttaa tcatatgcacttcagactac 7920 ctagcaagaa atcctaacca ttacatagaa ttggtagtga ttaattggtctctggtggtc 7980 cctgatttca gacacttagc ctgtgatgag gagagagtgg aagaagcaaaactgctggtg 8040 tcccaaggag caagtattta cattgagaat aaagaagaaa agacacccctgcaagtggcc 8100 aaaggtggcc tgggtttaat actcaagaga atggtggaag gttaaacagcttggatttat 8160 tcttactttg tatgttgtgt tgttgtcccc agtgtcctac aaactaatgtatttgtgcac 8220 aagacatcat ctatgaatga tgaagttttc tcaccttcaa agtcttataaacatgttgac 8280 tcttgttcct gctgagttac ttgttcgaag cttacagctt gttttccaggcatcgaataa 8340 ctgttgagat tgttctactg ttgtcgtata ttcttctata ttgaattctggttaatttgg 8400 agtaactaat tctgtggctg ttgtgagtct tcagcaccct cccatgtaccttatatccct 8460 ctctgaaaca gaacagctcc aatagcaaca agctagttgt tctgccagatgtttctatgt 8520 ggattctgta atgttcctcc atacagttaa aacatcctaa cttgtttttcaagctcactc 8580 aggcctacgc caaacgtttc tgtttttttt aaccatgagg tttaatttatttttgtgata 8640 ggagggatat ttacatattt tagtggacca cattttaagt tggatggtgtgctctaaaat 8700 actgaaaaac aatagcccat atacctatgt atttgttttt gatgggttgtttactctgaa 8760 ataaaatgta tggttttctt aaaaggaagt tttaaagtac ctattttgtgtcatcctgta 8820 ttgaaaagaa tgtcaagctt gttaaaatga catgtaacaa aaatgtattttgatttgtat 8880 ttcagaaact aaaaaataaa atgttgaaag aatcttccaa tttttctttcaaattagcta 8940 gaattcactt aagccttaga atccttagta aagacatttc tcagtttttggtagctctaa 9000 gtcaataaaa ttagtgaagc ttgaattgta gctaaaatct tttaaaatagccatcttctg 9060 aattgctaat taaatgtaga acaaaaggaa attattaagt ataaacttgagctccattta 9120 gggaagcatc ttaactgata ctagtcataa aatctaatga actccaagtgactttcagat 9180 tattttttat ttgaatttta tgctgcaaat actgctcgaa ataagtgatccaagtgaacc 9240 tacaaacttg cattaatttt cggtcaaagt cttaatgatc agtagctaaaatgagtttcg 9300 taaacacgaa ctgtgttact gatatagaaa ctttttacag aagtattgtgagtgctaacc 9360 ttaacaatca gagaagcatc ttatagaaca aaactctatt gaatgctggagatagacttg 9420 atagcagggg aaataatatt tctagtattt ttagtgtgat cagagtaaagttttgtctgt 9480 tacaactgaa gacatttact cttcataaat attgagttaa ttgtctgaggtgataggttt 9540 cgtgggtgta aaaggaaatt catccgagtt cttcaataag caatacagatggagctaagc 9600 cacaggtaat ttgtaggctt ttggcatttt aaatggcatg acttgttaatatttagatca 9660 cagatttgta taaaataaag atattagtgg cctcggatat tgatcagattgttacctgtg 9720 tctgtaggaa aggtgttaga gatgaagctg attcaaggtc agcagattttacagtttcaa 9780 aacataattt ggctgggcat ggtcgctcat gcctgttatc ccagcacattgggaggtcga 9840 ggcgggcaga ttacctgagg tcaggagttc aagaccagcc tgcccaacatggcgaaaccc 9900 catctctact aaaaaaatta gccgggtgtg gtggtgggcg cctgtaatcccagctactca 9960 ggaggttgag gcacgaaaat tgcttgaacc caggaggtgg aggttgcagtgagccgagtt 10020 ggtgccactg cagtctagcc tgggtgacag agcgacaatt catctcaaaatacataaata 10080 atttaatggt aaagcttaac tgagaatata cgatggaaga aaaaggccaatgaaaatagt 10140 gaaaacattt acattcttaa atatgttata gagaattaca atttcactgaggttttggga 10200 acttcaaagt aaatctgtgt ttttcttcat aatctttgac ttaacatttaactgtatgac 10260 actagtgttt gattccccaa tttctcagct gtaccaacta caatgaattccttagatggc 10320 tgcgtctgct gctttctgct cccatattct gttacactac aaggcctccaaatcaatctg 10380 ttggcaagtc tgatagagta taaagattct 10410 5 16 DNAArtificial Sequence PCR Primer 5 tgctggccgg gatgag 16 6 23 DNAArtificial Sequence PCR Primer 6 ccattttgat tgacagcatt cac 23 7 29 DNAArtificial Sequence PCR Probe 7 tgtaaaagcc cttctgggaa aaggtgctc 29 8 19DNA Artificial Sequence PCR Primer 8 gaaggtgaag gtcggagtc 19 9 20 DNAArtificial Sequence PCR Primer 9 gaagatggtg atgggatttc 20 10 20 DNAArtificial Sequence PCR Probe 10 caagcttccc gttctcagcc 20 11 1544 DNA H.sapiens CDS (99)...(779) 11 attggtgaag ctctaacggc tgttttgact ggcgtagccggagccggcga cgtgaggcgg 60 gcgttgctcg cgcgacaagt agttgctggg acagcgaa atggag ggg tgt gtg tct 116 Met Glu Gly Cys Val Ser 1 5 aac cta atg gtc tgcaac ctg gcc tac agc ggg aag ctg gaa gag ttg 164 Asn Leu Met Val Cys AsnLeu Ala Tyr Ser Gly Lys Leu Glu Glu Leu 10 15 20 aag gag agt att ctg gccgat aaa tcc ctg gct act aga act gac cag 212 Lys Glu Ser Ile Leu Ala AspLys Ser Leu Ala Thr Arg Thr Asp Gln 25 30 35 gac agc aga act gca ttg cactgg gca tgc tca gct gga cat aca gaa 260 Asp Ser Arg Thr Ala Leu His TrpAla Cys Ser Ala Gly His Thr Glu 40 45 50 att gtt gaa ttt ttg ttg caa cttgga gtg cca gtg aat gat aaa gac 308 Ile Val Glu Phe Leu Leu Gln Leu GlyVal Pro Val Asn Asp Lys Asp 55 60 65 70 gat gca ggt tgg tct cct ctt catatt gcg gct tct gct ggc cgg gat 356 Asp Ala Gly Trp Ser Pro Leu His IleAla Ala Ser Ala Gly Arg Asp 75 80 85 gag att gta aaa gcc ctt ctg gga aaaggt gct caa gtg aat gct gtc 404 Glu Ile Val Lys Ala Leu Leu Gly Lys GlyAla Gln Val Asn Ala Val 90 95 100 aat caa aat ggc tgt act ccc tta cattat gca gct tcg aaa aac agg 452 Asn Gln Asn Gly Cys Thr Pro Leu His TyrAla Ala Ser Lys Asn Arg 105 110 115 cat gag atc gct gtc atg tta ctg gaaggc ggg gct aat cca gat gct 500 His Glu Ile Ala Val Met Leu Leu Glu GlyGly Ala Asn Pro Asp Ala 120 125 130 aag gac cat tat gag gct aca gca atgcac cgg gca gca gcc aag ggt 548 Lys Asp His Tyr Glu Ala Thr Ala Met HisArg Ala Ala Ala Lys Gly 135 140 145 150 aac ttg aag atg att cat atc cttctg tac tac aaa gca tcc aca aac 596 Asn Leu Lys Met Ile His Ile Leu LeuTyr Tyr Lys Ala Ser Thr Asn 155 160 165 atc caa gac act gag ggt aac actcct cta cac tta gcc tgt gat gag 644 Ile Gln Asp Thr Glu Gly Asn Thr ProLeu His Leu Ala Cys Asp Glu 170 175 180 gag aga gtg gaa gaa gca aaa ctgctg gtg tcc caa gga gca agt att 692 Glu Arg Val Glu Glu Ala Lys Leu LeuVal Ser Gln Gly Ala Ser Ile 185 190 195 tac att gag aat aaa gaa gaa aagaca ccc ctg caa gtg gcc aaa ggt 740 Tyr Ile Glu Asn Lys Glu Glu Lys ThrPro Leu Gln Val Ala Lys Gly 200 205 210 ggc ctg ggt tta ata ctc aag agaatg gtg gaa ggt taa acagcttgga 789 Gly Leu Gly Leu Ile Leu Lys Arg MetVal Glu Gly 215 220 225 tttattctta ctttgtatgt tgtgttgttg tccccagtgtcctacaaact aatgtatttg 849 tgcacaagac atcatctatg aatgatgaag ttttctcaccttcaaagtct tataaacatg 909 ttgactcttg ttcctgctga gttacttgtt cgaagcttacagcttgtttt ccaggcatcg 969 aataactgtt gagattgttc tactgttgtc gtatattcttctatattgaa ttctggttaa 1029 tttggagtaa ctaattctgt ggctgttgtg agtcttcagcaccctcccat gtaccttata 1089 tccctctctg aaacagaaca gctccaatag caacaagctagttgttctgc cagatgtttc 1149 tatgtggatt ctgtaatgtt cctccataca gttaaaacatcctaacttgt ttttcaagct 1209 cactcaggcc tacgccaaac gtttctgttt tttttaaccatgaggtttaa tttatttttg 1269 tgataggagg gatatttaca tattttagtg gaccacattttaagttggat ggtgtgctct 1329 aaaatactga aaaacaatag cccatatacc tatgtatttgtttttgatgg gttgtttact 1389 ctgaaataaa atgtatggtt ttcttaaaag gaagttttaaagtacctatt ttgtgtcatc 1449 ctgtattgaa aagaatgtca agcttgttaa aatgacatgtaacaaaaatg tattttgatt 1509 tgtatttcag aaactaaaaa ataaaatgtt gaaag 154412 362 DNA H. sapiens unsure 322 unknown 12 gcgaaatgga ggggtgtgtgtctaacctaa tggtctgcaa cctggcctac agcgggaagc 60 tggaagagtt gaaggagagtattctggccg ataaatccct ggctactaga actgaccagg 120 caggttggtc tcctcttcatattgcggctt ctgctggccg ggatgagatt gtaaaagccc 180 ttctgggaaa aggtgctcaagtgaatgctg tcaatcaaaa tggctgtact cccttacatt 240 atgcagcttc gaaaaacaggcatgagatcg ctgtcatgtt actggaaggc ggggctaatc 300 cagatgctaa ggaccattatgnaggctaca gcaattgcac cgggcagcag ccaagggtta 360 ac 362 13 607 DNA H.sapiens 13 gggacagcga aatggagggg tgtgtgtcta acctaatggt ctgcaacctggcctacagcg 60 ggaagctgga agagttgaag gagagtattc tggccgataa atccctggctactagaactg 120 accaggacag cagaactgca ttgcactggg catgctcagc tggacatacagaaattgttg 180 aattttgttg caacttggag tgccagtgaa tgataaagac gatgcaggttggtctcctct 240 tcatattgcg gcttctgctg gccgggatga gattgtaaaa gcccttctgggaaaaggtgc 300 tcaagtgaat gctgtcaatc aaaatggctg tactccctta cattatgcagcttcgaaaaa 360 caggcatgag atcgctgtca tgttactgga aggcggggct aatccagatgctaaggacca 420 ttatgaggct acagcaatgc accgggcagc agccaaggac acttagcctgtgatgaggag 480 agagtggaag aagcaaaact gctggtgtcc caaggagcaa gtatttacattgagaataaa 540 gaagaaaaga cacccctgca agtggccaaa ggtggcctgg gtttaatactcaagagaatg 600 gtggaag 607 14 20 DNA Artificial Sequence AntisenseOligonucleotide 14 ccattctctt gagtattaaa 20 15 20 DNA ArtificialSequence Antisense Oligonucleotide 15 ctaaaatatg taaatatccc 20 16 20 DNAArtificial Sequence Antisense Oligonucleotide 16 tcccgctgta ggccaggttg20 17 20 DNA Artificial Sequence Antisense Oligonucleotide 17 acattttatttcagagtaaa 20 18 20 DNA Artificial Sequence Antisense Oligonucleotide 18actgtatgga ggaacattac 20 19 20 DNA Artificial Sequence AntisenseOligonucleotide 19 tagagcacac catccaactt 20 20 20 DNA ArtificialSequence Antisense Oligonucleotide 20 gctccggcta cgccagtcaa 20 21 20 DNAArtificial Sequence Antisense Oligonucleotide 21 ctttgtagta cagaaggata20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 tgctattggagctgttctgt 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23acctgcatcg tctttatcat 20 24 20 DNA Artificial Sequence AntisenseOligonucleotide 24 ggccaggttg cagaccatta 20 25 20 DNA ArtificialSequence Antisense Oligonucleotide 25 tattcgatgc ctggaaaaca 20 26 20 DNAArtificial Sequence Antisense Oligonucleotide 26 tcacttgagc accttttccc20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 gcgatctcatgcctgttttt 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28tcttgagtat taaacccagg 20 29 20 DNA Artificial Sequence AntisenseOligonucleotide 29 ttcaacaatt tctgtatgtc 20 30 20 DNA ArtificialSequence Antisense Oligonucleotide 30 atcggccaga atactctcct 20 31 20 DNAArtificial Sequence Antisense Oligonucleotide 31 cattcacttg agcacctttt20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 gtttttcgaagctgcataat 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33atgatgtctt gtgcacaaat 20 34 20 DNA Artificial Sequence AntisenseOligonucleotide 34 tcaacatgtt tataagactt 20 35 20 DNA ArtificialSequence Antisense Oligonucleotide 35 taggtacttt aaaacttcct 20 36 20 DNAArtificial Sequence Antisense Oligonucleotide 36 tttcgctgtc ccagcaacta20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 aacagttattcgatgcctgg 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38gcatgcccag tgcaatgcag 20 39 20 DNA Artificial Sequence AntisenseOligonucleotide 39 ttttaagaaa accatacatt 20 40 20 DNA ArtificialSequence Antisense Oligonucleotide 40 atgtcttgtg cacaaataca 20 41 20 DNAArtificial Sequence Antisense Oligonucleotide 41 ccagggattt atcggccaga20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 aacccaggccacctttggcc 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43tgagcatgcc cagtgcaatg 20 44 20 DNA Artificial Sequence AntisenseOligonucleotide 44 acttcatcat tcatagatga 20 45 20 DNA ArtificialSequence Antisense Oligonucleotide 45 ttcaagttac ccttggctgc 20 46 20 DNAArtificial Sequence Antisense Oligonucleotide 46 atcttcaagt tacccttggc20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 aaacagccgttagagcttca 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48ttgtggatgc tttgtagtac 20 49 20 DNA Artificial Sequence AntisenseOligonucleotide 49 atgcccagtg caatgcagtt 20 50 20 DNA ArtificialSequence Antisense Oligonucleotide 50 ttataagact ttgaaggtga 20 51 20 DNAArtificial Sequence Antisense Oligonucleotide 51 acacccctcc atttcgctgt20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 ggttagacacacacccctcc 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53ttctgctgtc ctggtcagtt 20 54 20 DNA Artificial Sequence AntisenseOligonucleotide 54 tggcactcca agttgcaaca 20 55 20 DNA ArtificialSequence Antisense Oligonucleotide 55 gccgcaatat gaagaggaga 20 56 20 DNAArtificial Sequence Antisense Oligonucleotide 56 ggagtacagc cattttgatt20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 cgccttccagtaacatgaca 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58atggtcctta gcatctggat 20 59 20 DNA Artificial Sequence AntisenseOligonucleotide 59 gcattgctgt agcctcataa 20 60 20 DNA ArtificialSequence Antisense Oligonucleotide 60 cttggctgct gcccggtgca 20 61 20 DNAArtificial Sequence Antisense Oligonucleotide 61 aggctaagtg tagaggagtg20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 cactctctcctcatcacagg 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63acaccagcag ttttgcttct 20 64 20 DNA Artificial Sequence AntisenseOligonucleotide 64 atgtaaatac ttgctccttg 20 65 20 DNA ArtificialSequence Antisense Oligonucleotide 65 ccaagctgtt taaccttcca 20 66 20 DNAArtificial Sequence Antisense Oligonucleotide 66 taagaataaa tccaagctgt20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 gggtgctgaagactcacaac 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68ttcagagagg gatataaggt 20 69 20 DNA Artificial Sequence AntisenseOligonucleotide 69 gcagaacaac tagcttgttg 20 70 20 DNA ArtificialSequence Antisense Oligonucleotide 70 taggatgttt taactgtatg 20 71 20 DNAArtificial Sequence Antisense Oligonucleotide 71 cttaaaatgt ggtccactaa20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 caagcttgacattcttttca 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73gtttctgaaa tacaaatcaa 20 74 20 DNA Artificial Sequence AntisenseOligonucleotide 74 gttgctttac ctggtcagtt 20 75 20 DNA ArtificialSequence Antisense Oligonucleotide 75 agcaccatgt aaacttctct 20 76 20 DNAArtificial Sequence Antisense Oligonucleotide 76 tttgtgggct tactaaaggc20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 gcagttggtttgtttaaatc 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78accaacctgc ctataaaaga 20 79 20 DNA Artificial Sequence AntisenseOligonucleotide 79 tgtcacttac agaggagtgt 20 80 20 DNA ArtificialSequence Antisense Oligonucleotide 80 taatactttt taaaagttgg 20 81 20 DNAArtificial Sequence Antisense Oligonucleotide 81 ggctaagtgt ctgaaatcag20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 accaacctgcctggtcagtt 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83ggctaagtgt ccttggctgc 20 84 20 DNA H. sapiens 84 tttaatactc aagagaatgg20 85 20 DNA H. sapiens 85 caacctggcc tacagcggga 20 86 20 DNA H. sapiens86 tatccttctg tactacaaag 20 87 20 DNA H. sapiens 87 acagaacagctccaatagca 20 88 20 DNA H. sapiens 88 taatggtctg caacctggcc 20 89 20 DNAH. sapiens 89 tgttttccag gcatcgaata 20 90 20 DNA H. sapiens 90gggaaaaggt gctcaagtga 20 91 20 DNA H. sapiens 91 aaaaacaggc atgagatcgc20 92 20 DNA H. sapiens 92 cctgggttta atactcaaga 20 93 20 DNA H. sapiens93 aggagagtat tctggccgat 20 94 20 DNA H. sapiens 94 aaaaggtgctcaagtgaatg 20 95 20 DNA H. sapiens 95 atttgtgcac aagacatcat 20 96 20 DNAH. sapiens 96 aagtcttata aacatgttga 20 97 20 DNA H. sapiens 97aggaagtttt aaagtaccta 20 98 20 DNA H. sapiens 98 tagttgctgg gacagcgaaa20 99 20 DNA H. sapiens 99 ccaggcatcg aataactgtt 20 100 20 DNA H.sapiens 100 tgtatttgtg cacaagacat 20 101 20 DNA H. sapiens 101tctggccgat aaatccctgg 20 102 20 DNA H. sapiens 102 ggccaaaggt ggcctgggtt20 103 20 DNA H. sapiens 103 cattgcactg ggcatgctca 20 104 20 DNA H.sapiens 104 tcatctatga atgatgaagt 20 105 20 DNA H. sapiens 105gcagccaagg gtaacttgaa 20 106 20 DNA H. sapiens 106 gccaagggta acttgaagat20 107 20 DNA H. sapiens 107 aactgcattg cactgggcat 20 108 20 DNA H.sapiens 108 tcaccttcaa agtcttataa 20 109 20 DNA H. sapiens 109acagcgaaat ggaggggtgt 20 110 20 DNA H. sapiens 110 ggaggggtgt gtgtctaacc20 111 20 DNA H. sapiens 111 aactgaccag gacagcagaa 20 112 20 DNA H.sapiens 112 tgttgcaact tggagtgcca 20 113 20 DNA H. sapiens 113tctcctcttc atattgcggc 20 114 20 DNA H. sapiens 114 aatcaaaatg gctgtactcc20 115 20 DNA H. sapiens 115 atccagatgc taaggaccat 20 116 20 DNA H.sapiens 116 ttatgaggct acagcaatgc 20 117 20 DNA H. sapiens 117tgcaccgggc agcagccaag 20 118 20 DNA H. sapiens 118 cctgtgatga ggagagagtg20 119 20 DNA H. sapiens 119 agaagcaaaa ctgctggtgt 20 120 20 DNA H.sapiens 120 caaggagcaa gtatttacat 20 121 20 DNA H. sapiens 121tggaaggtta aacagcttgg 20 122 20 DNA H. sapiens 122 acagcttgga tttattctta20 123 20 DNA H. sapiens 123 gttgtgagtc ttcagcaccc 20 124 20 DNA H.sapiens 124 accttatatc cctctctgaa 20 125 20 DNA H. sapiens 125caacaagcta gttgttctgc 20 126 20 DNA H. sapiens 126 catacagtta aaacatccta20 127 20 DNA H. sapiens 127 ttagtggacc acattttaag 20 128 20 DNA H.sapiens 128 tgaaaagaat gtcaagcttg 20 129 20 DNA H. sapiens 129ttgatttgta tttcagaaac 20 130 20 DNA H. sapiens 130 aactgaccag gtaaagcaac20 131 20 DNA H. sapiens 131 agagaagttt acatggtgct 20 132 20 DNA H.sapiens 132 gcctttagta agcccacaaa 20 133 20 DNA H. sapiens 133gatttaaaca aaccaactgc 20 134 20 DNA H. sapiens 134 acactcctct gtaagtgaca20 135 20 DNA H. sapiens 135 ctgatttcag acacttagcc 20

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targetedto a nucleic acid molecule encoding Gankyrin, wherein said compoundspecifically hybridizes with said nucleic acid molecule encodingGankyrin (SEQ ID NO: 4) and inhibits the expression of Gankyrin.
 2. Thecompound of claim 1 comprising 12 to 50 nucleobases in length.
 3. Thecompound of claim 2 comprising 15 to 30 nucleobases in length.
 4. Thecompound of claim 1 comprising an oligonucleotide.
 5. The compound ofclaim 4 comprising an antisense oligonucleotide.
 6. The compound ofclaim 4 comprising a DNA oligonucleotide.
 7. The compound of claim 4comprising an RNA oligonucleotide.
 8. The compound of claim 4 comprisinga chimeric oligonucleotide.
 9. The compound of claim 4 wherein at leasta portion of said compound hybridizes with RNA to form anoligonucleotide-RNA duplex.
 10. The compound of claim 1 having at least70% complementarity with a nucleic acid molecule encoding Gankyrin (SEQID NO: 4) said compound specifically hybridizing to and inhibiting theexpression of Gankyrin.
 11. The compound of claim 1 having at least 80%complementarity with a nucleic acid molecule encoding Gankyrin (SEQ IDNO: 4) said compound specifically hybridizing to and inhibiting theexpression of Gankyrin.
 12. The compound of claim 1 having at least 90%complementarity with a nucleic acid molecule encoding Gankyrin (SEQ IDNO: 4) said compound specifically hybridizing to and inhibiting theexpression of Gankyrin.
 13. The compound of claim 1 having at least 95%complementarity with a nucleic acid molecule encoding Gankyrin (SEQ IDNO: 4) said compound specifically hybridizing to and inhibiting theexpression of Gankyrin.
 14. The compound of claim 1 having at least onemodified internucleoside linkage, sugar moiety, or nucleobase.
 15. Thecompound of claim 1 having at least one 2′-O-methoxyethyl sugar moiety.16. The compound of claim 1 having at least one phosphorothioateinternucleoside linkage.
 17. The compound of claim 1 having at least one5-methylcytosine.
 18. A method of inhibiting the expression of Gankyrinin cells or tissues comprising contacting said cells or tissues with thecompound of claim 1 so that expression of Gankyrin is inhibited.
 19. Amethod of screening for a modulator of Gankyrin, the method comprisingthe steps of: a. contacting a preferred target segment of a nucleic acidmolecule encoding Gankyrin with one or more candidate modulators ofGankyrin, and b. identifying one or more modulators of Gankyrinexpression which modulate the expression of Gankyrin.
 20. The method ofclaim 19 wherein the modulator of Gankyrin expression comprises anoligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, anRNA oligonucleotide, an RNA oligonucleotide having at least a portion ofsaid RNA oligonucleotide capable of hybridizing with RNA to form anoligonucleotide-RNA duplex, or a chimeric oligonucleotide.
 21. Adiagnostic method for identifying a disease state comprising identifyingthe presence of Gankyrin in a sample using at least one of the primerscomprising SEQ ID NOs 5 or 6, or the probe comprising SEQ ID NO:
 7. 22.A kit or assay device comprising the compound of claim
 1. 23. A methodof treating an animal having a disease or condition associated withGankyrin comprising administering to said animal a therapeutically orprophylactically effective amount of the compound of claim 1 so thatexpression of Gankyrin is inhibited.
 24. The method of claim 23 whereinthe disease or condition is a hyperproliferative disorder.